This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2000-060578, filed Mar. 6, 2000, the entire contents of which are incorporated herein by reference.
1. Field of the Invention
The present invention relates to a pattern formation member applied to a sectioning image observation apparatus for observing/measuring sample microstructure or three-dimensional shape of a sample by using light and a sectioning image observation apparatus using them.
2. Description of the Related Art
Conventionally, as a sectioning image observation apparatus, a confocal microscope using a rotation disk called Nipkow rotation disk where a number of pin holes are arranged in spiral with an interval of about ten times of the pin hole diameter is known.
FIG. 1 shows the schematic configuration of a confocal microscope using such a Nipkow rotation disk, wherein a condenser lens 2 and a PBS (polarized beam splitter) 3 are arranged on a light path of the light emitted from a light source 1 such as halogen light source or mercury light source or others, and a Nipkow rotation disk (called rotation disk, hereinafter) 4, a first imaging lens 5, xc2xc wavelength plate 6 and a sample 8 through an objective 7 are arranged on the reflected light path of the PBS 3. In addition, a CCD camera 10 is arranged through a second imaging lens 9 on the filtered light path of the PBS 3 of the light reflected from the sample 8. A monitor 11 is connected to the image output terminal of this CCD camera 10 for displaying the image taken by the CCD camera.
Here, pin holes 4a are arranged in spiral on the rotation disk 4 with an interval of about ten times of the pin hole diameter between respective pin holes, and the rotation disk 4a is connected to the shaft of a not shown motor via a rotation shaft 12 and rotated at a fixed rotation speed.
In such configuration, the light emitted from the light source 1 passes through the condenser lens 2 and only polarized component of a fixed direction is reflected by the PBS 3 and input to the rotation disk 4 rotating at the fixed speed, and the light filtered by the pin hole 4a of this rotation disk 4 passes through the first imaging lens 5, circularly polarized by the xc2xc wavelength plate 6, imaged by the objective lens 7 and input to the sample 8. On the other hand, the light reflected from the sample 8 passes through the objective lens 7, takes a polarization direction orthogonal to the incident light again at the xc2xc wavelength plate 6, and projects the sample image on the rotation disk 4 by means of the first imaging lens 5. A focused portion of the sample image projected on the rotation disk 4 passes through the pin hole 4a, further passes through the PBS 3 and taken by the CCD camera 10 through the second imaging lens 9. A confocal image taken by the CCD camera 10 is displayed on the monitor 11.
Such confocal microscope allows to observe a so-called sectioning image, namely image for each level of the sample 8, by moving the focus vertically (Z axis direction), as only images having focused position (height) where the pin hole 4a of the rotation disk 4 passes can be observed.
By the way, for the confocal microscope using such Nipkow rotation disk, it is necessary to dispose pin holes on the rotation disk so that unevenness may not come into prominence in the observation field during the eye observation or imaging by a CCD camera. In short, it is necessary to arrange pin holes so that the sample observation field is illuminated evenly within a human perceptible time interval (about {fraction (1/20)} to {fraction (1/30)} sec) or CCD camera exposure time (often {fraction (1/60)} or {fraction (1/30)} sec).
Therefore, conventionally, various proposals have been made concerning the pin hole arrangement and, for instance, an arrangement wherein a plurality of pin holes are arranged in spiral in the rotation disk radial direction with an equal angle is known as the simplest arrangement. However, in such pin hole arrangement, the brightness of captured image is uneven, because the pin hole pitch is different in the outer circumferential section and the inner circumferential section of the rotation disk.
As a method to solve such problem, various pin hole arrangements for reducing the uneven brightness of captured image, such as an arrangement wherein the radial pitch of the locus of the virtual center line connecting centers of a plurality of pin holes composing pin hole lines arranged in spiral and the circumferential pitch along the spiral are made equal, or an arrangement wherein all pin holes composing a plurality of pin hole lines are differentiated in diameter at their center position have been proposed.
However, in the former pin hole arrangement, certainly, the image brightness in the observation field is even when the rotation disk center and the rotation axis agree exactly, but the observed image brightness is uneven when the rotation disk center and the rotation axis disagree. In general, the pin hole diameter is so small as about several dozens of xcexcm (45 xcexcm for 100 times, 100 xcexcm for 250 times); therefore, it is necessary to limit the difference between the rotation disk center and the rotation center to 10 xcexcm or less, namely sufficiently smaller than the pin hole diameter so that the observed image brightness may not be uneven, thereby, requiring an extremely high precision for perforation of pin hole on the rotation disk, shaping of the rotation disk, attachment of the rotation disk to the rotation shaft, or other processing.
On the other hand, the latter pin hole arrangement is improved to reduce the unevenness of observed image brightness; however, the unevenness is certainly reduced, but not eliminated.
In addition, when pin holes are formed on the rotation disk in this way, the pin hole arrangement is so devised not to make the observed image brightness uneven for all samples, and the pin hole is positioned using a complicated pattern prepared extremely precisely, in order to position each pin hole exactly. For instance, for Nipkow rotation disk, Cr or low-reflective Cr film is formed on a glass substrate, masked with a pin hole pattern and etched, and this mask is prepared by a EB drawing machine using electron beam similarly as semiconductor manufacturing, making the rotation disk preparation very costly and expensive due to the use of such complicated pattern mask.
Therefore, in order to solve these problems, it has been proposed a rotation disk wherein a straight line pattern section 141 including linearly formed translucent sections and shield sections arranged alternately, a full translucent section 142, and shield sections 143, 144 in each fan-shaped areas between these straight line pattern section 141 and full translucent section 142 are disposed on a rotation disk 14 as shown in FIG. 3A, and the width of translucent sections and shield sections of the straight line pattern section 141 among them is set to about several dozens of xcexcm similarly as the pin hole diameter, and formed to 1:1 as shown in FIG. 3A and FIG. 3B.
According to such rotation disk, first, an observation when the observation field passes through the straight line pattern section 141 is taken by the CCD camera, then an observation when it passes through the full translucent section 142 is taken by the CCD camera. In this case, a combined image (confocal image including non-confocal component) including not only an image having focused position (height) components (confocal component), but also image having non-focused position (height) components (permeated non-confocal component) is obtained, because the ratio of each width of translucent sections 141a and shield sections 141b is equal, for the image taken in the straight line pattern section 141. Consequently, only the confocal image having position (height) components in good focus ban be obtained by the difference calculation of bright-field taken through the full translucent section 142 from this combined image. In addition, uneven brightness is not generated in the observation image even when the rotation disk rotation center has shifted, and the rotation disk preparation cost will be limited because the pattern for creating the straight line pattern section 141 including linearly formed translucent sections and shield sections arranged alternately is a simple linearly pattern.
On the contrary, in the rotation disk 141 shown in FIG. 3A and FIG. 3B, the non-confocal component is prominent, because the ratio of each width of translucent sections and shield sections of the straight line pattern section 141 is 1:1. Therefore, a so-called sectioning effect, containing only confocal image can be expected only by the difference calculation. This generates problems such as impossibility of directly viewing the confocal image, necessity of operation equipment such as computer for image processing, enlargement of equipment scale, cost increase, and moreover, two images subjected to the difference calculation are susceptible to disturbance such as vibration, because they are taken with different timing.
An object of the present invention is to provide a pattern formation member applied to a sectioning image observation apparatus for stably observing a good image, without making the observed image brightness uneven and a sectioning image observation apparatus.
A pattern formation member adopted to a sectioning image observation apparatus which selectively irradiates a light from a light source to a sample, scans the sample, and acquires a light from the sample as a sectioning image, is characterized in that the pattern formation member comprises an irradiation section and a cutoff section, each of the irradiation section and the cutoff section is in a straight pattern, and these straight patterns are disposed alternatively.
Another pattern formation member adopted to a sectioning image observation apparatus which has a rotation disk having a translucent section which passes a light and a shield section which shields a light and rotating on a light path, irradiates a light passing through the translucent section to a sample, scans the sample, and passes a light from the sample passed through the rotation disk to acquire a sectioning image, is characterized in that each of patterns to scan the sample by the light passing through the rotation disk is formed in a straight pattern, and these patterns are disposed alternatively, straight pattern areas of the translucent section and the shield section with different direction are formed not to be parallel to a scanning direction (H direction) according to a rotation of the rotation disk in an observation field.
Preferable manners of the present invention are as follows.
(1) The pattern formation member is a rotation disk such that the irradiation section is a translucent section to pass a light and the cutoff section is a shield section to shield a light, the rotation disk is rotated on a light path, each of patterns to scan the sample by the light passing through the rotation disk is formed in a straight pattern, and these patterns are disposed alternatively.
(2) A shield area is formed at a portion to which straight patterns of the translucent section and the shield section of the rotation disk is parallel to a scanning direction (H direction) according to a rotation of the rotation disk in an observation field.
(3) The straight pattern areas have a plurality of sector shaped areas divided in a circumferential direction of the rotation disk.
(4) A portion parallel to a scanning direction (H direction) according to a rotation of the rotation disk in an observation field has another straight pattern area of the translucent section and the shield section with sector shape having a predetermined central angel whose direction differs from the straight pattern.
(5) A width of the straight pattern of the shield section is larger than that of the translucent section.
(6) The pattern formation member is a digital micro mirror having a plurality of mirrors, whose directions are independently changeable, disposed in a two-dimensional form.
(7) A plurality of areas having different ratios of the translucent section and the shield section are further provided.
(8) A plurality of areas having different direction of the translucent section and the shield section of the straight pattern of the rotation disk are further provided.
(9) The rotation disk is a rotation disk in which a rotation radial direction of the rotation disk is not normal to a direction of the straight pattern of the translucent section and shield section.
(10) A width of a straight portion of the rotation disk which shield a light is larger than a width of a straight portion thereof which passes a light.
(11) A width of a straight portion of the rotation disk is substantially constant.
(12) The rotation disk is divided into a plurality of areas and a pattern of each of the plurality of areas is different.
(13) A pattern of each of the plurality of areas has an equal area ratio of the translucent section and the shield section, and widths of the translucent section and the shield section are different for each of the areas.
(14) When a width of different direction area having a constant width is X and a period of the translucent section and the shield section is W in the rotation disk, X/W is constant.
(15) The patterns of the plurality of concentric circle areas have an equal area ratio of the translucent section and the shield section, a width of inner circumference concentric circle area is smaller than that of outer circumference concentric circle area, and a width of different direction area of the inner circumference concentric circle area is smaller than that of outer circumference concentric circle area.
(16) When the translucent sections of the least two concentric circle areas have a same width and a period W of the translucent section and the shield section is different, a period of the translucent section and the shield section on an inner concentric circle area is smaller than that of an outer concentric circle area, and a width X of a different direction area of inner and outer concentric circle areas is proportional to the period W.
A sectioning image observation apparatus according to the present invention scans a sample with a light by using any one of above-mentioned pattern formation members, and acquires a reflected light from the sample as a sectioning image through the pattern formation member. With this arrangement, it is preferable that a moving mechanism to change a projection position on the rotation disk to the sample is further provided.
Another sectioning image observation apparatus according to the present invention enters an excited light with a predetermined wavelength through an excitation filter to any one of above-mentioned pattern formation members, scans a sample with a light by using the pattern formation member, and acquires a fluorescence emitted from the sample as a sectioning image through the pattern formation member and a barrier filter selecting a wavelength of the emitted fluorescence.
A still another sectioning image observation apparatus is characterized by comprising: a light source; a rotation disk having a pattern in which a slit translucent section which passes a light and a straight shading section which shields a light, are alternately and periodically arranged; means to lead a light from the light source to the rotation disk; means to irradiate a light passing the rotation disk to a sample and project a pattern of the rotation disk to the sample; an optical lens which projects a light reflected from the sample on the rotation disk; and means to rotate the rotation disk on an optical path, scan the pattern of the rotation disk projected on the sample, and acquires an image passing the rotation disk as an sectioning image among sample images projected on the rotation disk, and when an angle of the rotation disk surface and a surface normal to an optical axis is xcex8, an aperture of the lens from the sample is NA, an expansion rate of a sample image projected on the rotation disk is M, a diameter (called as a number of view) on the rotation disk in an area of the observed sample is R, an angle between a main light beam which passes at an outermost edge of a diameter on the rotation disk of the observed sample area and an optical axis is xcfx86, and a wavelength of the light is xcex, at least one of the following conditions are satisfied:
xcex8 greater than xcfx862NA/M, and   θ   less than                               M          2                ⁢        λ                              NA          2                ⁢        R              .  
As the result, according to the present invention, a high quality observation image without uneven brightness can be obtained even when the rotation disk rotation center has shifted, because the straight pattern of translucent sections and shield sections are scanned while changing the direction thereof according to the rotation of the rotation disk 141.
Also, uneven brightness is prevented from occurring in the observed image, because it is so devised that the scanning direction (H direction) by the rotation of the rotation disk in the observation field and the direction of the straight pattern of translucent sections and shield sections will not be parallel.
Moreover, the mask pattern preparation is simple and cheap in cost, because the straight patterns of translucent sections and shield sections are only arranged alternately.
In addition, according to the present invention, the permeability of the rotation disk can be set by providing a plurality of areas where a pattern constituted of alternately disposed straight translucent sections and shield sections, changing the line width for each area, and allowing to move the rotation disk use area, the sectioning effect and the image brightness can be set selectively according to the sample situation, light can be used effectively according to the sample, and it becomes possible to obtain a bright sectioning image for various kinds of samples.
Further, according to the present invention, a pattern corresponding to the objective magnification or number of apertures, among a plurality of patterns on the disk, without making the observed image brightness uneven, so a disk applied to a sectioning image observation apparatus for stably observing a good image, and a sectioning image observation apparatus can be supplied.
Besides, according to the present invention, a confocal image can be observed even with a plurality of objectives, and images of different confocal effect can be observed, by dividing a disk where translucent sections and shield sections are arranged linearly into a plurality of concentric areas, and changing the translucent section slit width (L) and the shield section width (W-L) in each area, and at the same time, every confocal image observed in any area can be made homogenous and satisfactory, because the width X of a different direction area where patterns for suppressing the generation of alternating contrast stripes can be decided by the cycle W of translucent sections and shield sections. Further, as the width of different direction area can be decided easily, it is unnecessary to remake times and times for deciding the width of this area, reducing the examination time and the cost.
Moreover, according to the present invention, the rotation disk inclination angle can be decided practically for reducing unnecessary reflected light (flare) by calculation considering the magnification of the sample image projected on the disk, field of view range, and light incident angle; therefore, not only the angle can be decided to obtain a good contrast sectioning image free of flare, but also it is possible to include the disk inclination within the focal depth of the sample, preventing an image focused to different height on the sample from being observed.
Still further, according to the present invention, in place of scanning the pattern where straight translucent sections and shield sections are arranged alternately using a disk, the pattern is created and scanned by using a micro mirror array and changing the direction of respective micro mirror. Consequently, the slit light width can be created in correspondence to various objectives, making useless to exchange disks, or make a disk divided into a plurality of areas circumferentially, and a quality confocal image can be obtained simply, as a pattern corresponding to an objective can be created, without modification.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.